Sunitinib formulations and methods for use thereof in treatment of glaucoma

ABSTRACT

Methods for increasing the encapsulation or incorporation of Sunitinib into polymeric matrices have been developed. The resulting formulations provide for more sustained controlled release of sunitinib or other inhibitors of JNK signaling, which bind to DLK. Increased loading is achieved using an alkaline solvent system. The pharmaceutical compositions can be administered to treat or reduce neuronal death due to elevated intraocular pressure. Upon administration, the sunitinib or other inhibitor is released over an extended period of time at concentrations which are high enough to produce therapeutic benefit, but low enough to avoid unacceptable levels of cytotoxicity, and which provide much longer release than inhibitor without conjugate.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 16/692,691, filedNov. 22, 2019, which is a continuation of U.S. Ser. No. 15/536,272,filed Jun. 15, 2017, entitled “SUNITINIB FORMULATIONS AND METHODS FORUSE THEREOF IN TREATMENT OF GLAUCOMA”, which issued on Jan. 7, 2020 asU.S. Pat. No. 10,525,034, and which is a National Phase applicationunder 35 U.S.C. § 371 of International Application No.PCT/US2015/065878, filed Dec. 15, 2015, which claims priority to andbenefit of U.S. Provisional Application No. 62/092,118 “ControlledRelease Sunitinib Formulations” filed on Dec. 15, 2014, and U.S.Provisional Application No. 62/139,306 “Method Of Prevention Of CornealNeovascularization” filed Mar. 27, 2015, the disclosures of which arehereby incorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under EY023754 andEY026578 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted Dec. 15, 2015 as a text file named“JHU_C13492_PCT_ST25” created on Dec. 15, 2015, and having a size of 746bytes is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to sunitinib formulations and methods ofuse thereof, especially for use in the treatment of ocular diseases andother neuronal disorders.

BACKGROUND OF THE INVENTION

There are several types of glaucoma. The two main types are open-angleand angle-closure. These are marked by an increase of intraocularpressure (IOP), or pressure inside the eye.

Open-angle glaucoma, the most common form of glaucoma, accounting for atleast 90% of all glaucoma cases, is caused by the slow clogging of thedrainage canals, resulting in increased eye pressure. It has a wide andopen angle between the iris and cornea, develops slowly and is alifelong condition, and has symptoms and damage that are not noticed.“Open-angle” means that the angle where the iris meets the cornea is aswide and open as it should be. Open-angle glaucoma is also calledprimary or chronic glaucoma. It is the most common type of glaucoma,affecting about three million Americans.

Angle-closure glaucoma, a less common form of glaucoma: It is caused byblocked drainage canals, resulting in a sudden rise in intraocularpressure, has a closed or narrow angle between the iris and cornea,develops very quickly, has symptoms and damage that are usually verynoticeable, and demands immediate medical attention. It is also calledacute glaucoma or narrow-angle glaucoma. Unlike open-angle glaucoma,angle-closure glaucoma is a result of the angle between the iris andcornea closing.

Normal-Tension Glaucoma (NTG) or low-tension or normal-pressure glaucomais where the optic nerve is damaged even though the eye pressure is notvery high. Congenital Glaucoma occurs in babies when there is incorrector incomplete development of the eye's drainage canals during theprenatal period. This is a rare condition that may be inherited. Whenuncomplicated, microsurgery can often correct the structural defects.Other cases are treated with medication and surgery. Other Types ofGlaucoma include: Secondary Glaucoma, Pigmentary Glaucoma,Pseudoexfoliative Glaucoma, Traumatic Glaucoma, Neovascular Glaucoma,Irido Corneal Endothelial Syndrome (ICE), and Uveitic Glaucoma.

Open angle glaucoma is the most common form of glaucoma, affecting aboutthree million Americans. It happens when the eye's drainage canalsbecome clogged over time. The inner eye pressure (intraocular pressureor IOP) rises because the correct amount of fluid cannot drain out ofthe eye. With open-angle glaucoma, the entrances to the drainage canalsare clear and should be working correctly. If open-angle glaucoma is notdiagnosed and treated, it can cause a gradual loss of vision. This typeof glaucoma develops slowly and sometimes without noticeable sight lossfor many years. It usually responds well to medication, especially ifcaught early and treated.

Vision loss in glaucoma, a neurodegenerative disease that is the leadingcause of irreversible blindness worldwide, is due to the dysfunction anddeath of retinal ganglion cells (RGCs). Current therapies all act bylowering intraocular pressure (IOP). However, pressure reduction can bedifficult to achieve, and even with significant pressure lowering, RGCloss can continue. Efforts have therefore been made to developneuroprotective agents that would complement IOP-lowering by directlyinhibiting the RGC cell death process, though no neuroprotective agentis yet in clinical use.

It is an object of the invention to provide methods for encapsulation orincorporation into polymeric matrices, including nano- andmicro-particles, with increased loading, of drugs for treatment ofglaucoma and neuronal damage.

It is still another object of the invention to provide improved dosageformulations, prolonged pharmacokinetics, and methods of use thereof.

SUMMARY OF THE INVENTION

Methods for increasing the encapsulation or incorporation of Sunitinibinto polymeric matrices have been developed. The resulting formulationsprovide for more sustained controlled release of sunitinib for reductionor prevention of neuronal death due to elevated intraocular pressureassociated with glaucoma. Increased loading is achieved using analkaline solvent system.

Examples demonstrate that polyesters such as PEG-PLGA(PLA) andPEG-PLGA/blend microparticles display sustained release of sunitinib.Polymer microparticles composed of PLGA and PEG covalently conjugated toPLGA (M_(w) 45 kDa) (PLGA45k-PEG5k) loaded with Sunitinib were preparedusing a single emulsion solvent evaporation method. Maximum loading wasachieved by increasing the alkalinity of sunitinib, up to 16.1% withPEG-PLGA, which could be further increased by adding DMF, compared toonly 1% with no alkaline added.

The drugs can be used to form implants (e.g., rods, discs, wafers,etc.), nanoparticles, or microparticles with improved properties forcontrolled delivery of drugs. Pharmaceutical compositions containingimplants (e.g., rods, discs, wafers, etc.), nanoparticles,microparticles, or combinations thereof for the controlled release ofthe Sunitinib can be prepared by combining the drug in the matrix withwith one or more pharmaceutically acceptable excipients. Thenanoparticles, microparticles, or combination thereof can be formed fromone or more drugs, or blends of drugs with one or more polymers.

It has been discovered that dual-leucine zipper kinase (DLK) is a keyneuroprotective drug target of sunitinib. Supporting this finding, anumber of other neuroprotective kinase inhibitors also inhibit DLK.Sustained controlled release formulations of these compounds can beadministered to treat or reduce neuronal death due to elevatedintraocular pressure. Examples demonstrate in animal models that thesunitinib formulations are efficacious in preventing optic nerve damagedue to elevated intraocular pressure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph showing sunitinib promotes RGC survival in vitro andin vivo, survival of immunopanned RGCs, treated with increasing doses ofsunitinib, after 72 hours in culture. FIG. 1B graphs percent survival ofRGCs after optic nerve transection in rats pretreated with intravitrealdrug-eluting microspheres containing vehicle (n=10), 440 ng sunitinib(n=6) or 300 ng SR8165 (n=10). FIG. 1C is a graph showing optic nerveaxon counts following laser-induced ocular hypertension in ratspretreated with intravitreal microspheres containing vehicle (n=29), 440ng sunitinib (n=8) or 100 ng (n=24), 300 ng (n=26) or 600 ng (n=25)SR8165.

FIG. 2A is a schematic of laser induced ocular hypertension in a ratglaucoma model. FIG. 2B is a graph of Mean IOP increase 24 hours afterthe first administration of diode laser to the trabecular meshwork. FIG.2C is a graph of IOP 24 hours after the first administration of diodelaser, divided by treatment group.

FIG. 3A is a graph of viable cells showing survival of immunopannedRGCs, treated with increasing doses of SR8165, after 72 hours inculture. The most efficacious doses of sunitinib are shown forcomparison. FIGS. 3B-3F are graphs of viable cells, showing the lack ofneuroprotective activity of kinase inhibitors targeting VEGFR2, c-Kit,FLT3 and PDGFRs, of immunopanned RGCs, treated with increasing doses ofthe various kinase inhibitors, after 72 hours in culture.

FIG. 4A is a graph of knockdown of DLK mRNA and protein by DLK siRNA.RGCs were transfected with DLK or a nontargeting control (NT) siRNA.mRNA levels were quantified at 24 hours using RT-PCR.

FIG. 4B is a graph showing survival of immunopanned RGCs transfectedwith control (dashed) or DLK siRNA (solid). FIG. 4C is a graph ofpercent survival of RGCs 10 days after optic nerve crush in Dlk^(fl/fl)mice (n=3), Dlk^(fl/fl) ^(a)mice injected with AAV2-_(Cre) (n=8) orDlk^(+/+) mice injected with AAV2-Cre (n=9), normalized to uninjuredcontrol mice (n=6). FIG. 4D are the patch-clamp recordings from RGCsmaintained with DLK siRNA and/or sunitinib in response to depolarizingcurrent (left) or glutamate iontophoresis (right). *p<0.05, #p<0.005;error bars, s.d.

FIG. 5A is a graph showing DLK protein is upregulated in RGCs inresponse to injury, showing levels of DLK mRNA normalized to GAPDH,after various times in culture. FIG. 5B is a graph of survival, measuredby CellTiter-Glo (CTG) luminescence, of immunopanned RGCs 48 hours aftertransduction with adenovirus (MOI 1000) expressing wildtype (WT) orkinase-dead (KD) DLK. *p<0.05; error bars, s.d.

FIGS. 6A-6G are graphs of survival of immunopanned RGCs, treated withincreasing doses of the indicated DLK inhibitors: foretinib,lestaurtinib, tozasertib, crizotinib, KW-2449, axitinib, and bosutinib,after 72 hours in culture. FIG. 6H is a graph of the relationshipbetween the biochemical K_(d) (ability of the inhibitor to bind purifiedDLK) and the cellular ED₅₀. FIG. 6I is a graph of survival showingTozasertib protects RGC axons from glaucomatous injury in rats. FIG. 6Jis a graph of optic nerve axon counts following laser-induced ocularhypertension in rats pretreated with intravitreal drug-elutingmicrospheres containing vehicle (n=29), 82 ng (n=22) or 275 ng (n=21)tozasertib. Fellow eyes (n=157) shown for comparison. #p<0.005; errorbars, s.d.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

“Active Agent,” as used herein, refers to a physiologically orpharmacologically active substance that acts locally and/or systemicallyin the body. An active agent is a substance that is administered to apatient for the treatment (e.g., therapeutic agent), prevention (e.g.,prophylactic agent), or diagnosis (e.g., diagnostic agent) of a diseaseor disorder. “Ophthalmic Drug” or “Ophthalmic Active Agent”, as usedherein, refers to an agent that is administered to a patient toalleviate, delay onset of, or prevent one or more symptoms of a diseaseor disorder of the eye, or diagnostic agent useful for imaging orotherwise assessing the eye.

“Effective amount” or “therapeutically effective amount,” as usedherein, refers to an amount of drug effective to alleviate, delay onsetof, or prevent one or more symptoms, particularly of cancer or a diseaseor disorder of the eye. In the case of age-related macular degeneration,the effective amount of the drug delays, reduces, or prevents visionloss in a patient.

As used herein, the term “alkaline” refers to a compound capable ofaccepting an acidic proton or otherwise raising the pH of thecomposition.

“Biocompatible” and “biologically compatible,” as used herein, generallyrefer to materials that are, along with any metabolites or degradationproducts thereof, generally non-toxic to the recipient, and do not causeany significant adverse effects to the recipient. Generally speaking,biocompatible materials are materials which do not elicit a significantinflammatory or immune response when administered to a patient.

“Biodegradable Polymer,” as used herein, generally refers to a polymerthat will degrade or erode by enzymatic action and/or hydrolysis underphysiologic conditions to smaller units or chemical species that arecapable of being metabolized, eliminated, or excreted by the subject.The degradation time is a function of polymer composition, morphology,such as porosity, particle dimensions, and environment.

“Hydrophilic,” as used herein, refers to the property of having affinityfor water. For example, hydrophilic polymers (or hydrophilic polymers)are polymers (or polymers) which are primarily soluble in aqueoussolutions and/or have a tendency to absorb water. In general, the morehydrophilic a polymer is, the more that polymer tends to dissolve in,mix with, or be wetted by water.

“Hydrophobic,” as used herein, refers to the property of lackingaffinity for, or even repelling water. For example, the more hydrophobica polymer (or polymer), the more that polymer (or polymer) tends to notdissolve in, not mix with, or not be wetted by water.

Hydrophilicity and hydrophobicity can be spoken of in relative terms,such as, but not limited to, a spectrum of hydrophilicity/hydrophobicitywithin a group of polymers or polymers. In some embodiments wherein twoor more polymers are being discussed, the term “hydrophobic polymer” canbe defined based on the polymer's relative hydrophobicity when comparedto another, more hydrophilic polymer.

“Microparticle,” as used herein, generally refers to a particle having adiameter, such as an average diameter, from about 1 micron to about 100microns, preferably from about 1 micron to about 50 microns, morepreferably from about 1 to about 30 microns. The microparticles can haveany shape. Microparticles having a spherical shape are generallyreferred to as “microspheres”.

“Molecular weight,” as used herein, generally refers to the relativeaverage chain length of the bulk polymer, unless otherwise specified. Inpractice, molecular weight can be estimated or characterized usingvarious methods including gel permeation chromatography (GPC) orcapillary viscometry. GPC molecular weights are reported as theweight-average molecular weight (Mw) as opposed to the number-averagemolecular weight (Mn). Capillary viscometry provides estimates ofmolecular weight as the inherent viscosity determined from a dilutepolymer solution using a particular set of concentration, temperature,and solvent conditions.

“Mean particle size,” as used herein, generally refers to thestatistical mean particle size (diameter) of the particles in apopulation of particles. The diameter of an essentially sphericalparticle may refer to the physical or hydrodynamic diameter. Thediameter of a non-spherical particle may refer preferentially to thehydrodynamic diameter. As used herein, the diameter of a non-sphericalparticle may refer to the largest linear distance between two points onthe surface of the particle. Mean particle size can be measured usingmethods known in the art, such as dynamic light scattering.

“Monodisperse” and “homogeneous size distribution” are usedinterchangeably herein and describe a population of nanoparticles ormicroparticles where all of the particles are the same or nearly thesame size. As used herein, a monodisperse distribution refers toparticle distributions in which 90% or more of the distribution lieswithin 15% of the median particle size, more preferably within 10% ofthe median particle size, most preferably within 5% of the medianparticle size.

“Pharmaceutically Acceptable,” as used herein, refers to compounds,carriers, excipients, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

“Implant,” as generally used herein, refers to a polymeric device orelement that is structured, sized, or otherwise configured to beimplanted, preferably by injection or surgical implantation, in aspecific region of the body so as to provide therapeutic benefit byreleasing one or more active agents over an extended period of time atthe site of implantation. For example, intraocular implants arepolymeric devices or elements that are structured, sized, or otherwiseconfigured to be placed in the eye, preferably by injection or surgicalimplantation, and to treat one or more diseases or disorders of the eyeby releasing one or more drugs over an extended period. Intraocularimplants are generally biocompatible with physiological conditions of aneye and do not cause adverse side effects. Generally, intraocularimplants may be placed in an eye without disrupting vision of the eye.

II. Compositions A. DLK Inhibitors

It has been discovered that dual-leucine zipper kinase (DLK) is a keyneuroprotective drug target of sunitinib. Supporting this finding, anumber of other neuroprotective kinase inhibitors also inhibit DLK.These include SR8165, axitinib, bosutinib, neratininb, crizotinib,tozasertib, lestautinib, foretinib, TAE-684 and KW-2449

Sustained controlled release formulations of these compounds can beadministered to treat or reduce neuronal death due to elevatedintraocular pressure.

It has been suggested that sunitinib may be useful for treatment ofglaucoma. Sunitinib (marketed as SUTENT® by Pfizer, and previously knownas SU11248) is an oral, small-molecule, multi-targeted receptor tyrosinekinase (RTK) inhibitor that was approved by the FDA for the treatment ofrenal cell carcinoma (RCC) and imatinib-resistant gastrointestinalstromal tumor (GIST) on Jan. 26, 2006. Sunitinib was the first cancerdrug simultaneously approved for two different indications.

Sunitinib inhibits cellular signaling by targeting multiple receptortyrosine kinases (RTKs).These include all receptors for platelet-derivedgrowth factor (PDGF-Rs) and vascular endothelial growth factor receptors(VEGFRs), which play a role in both tumor angiogenesis and tumor cellproliferation. The simultaneous inhibition of these targets leads toboth reduced tumor vascularization and cancer cell death, and,ultimately, tumor shrinkage. Sunitinib is also neuroprotective by virtueof its kinase inhibitory activity.

Sunitinib is a compound of formula (1):

wherein

R¹ is selected from the group consisting of hydrogen, halo, alkyl,cyclkoalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy,—(CO)R¹⁵, —NR¹³R¹⁴, —(CH₂)_(r)R¹⁶ and —C(O)NR⁸R⁹;

R² is selected from the group consisting of hydrogen, halo, alkyl,trihalomethyl, hydroxy, alkoxy, cyano, —NR¹³R¹⁴, —NR¹³C(O)R¹⁴, —C(O)R¹⁵,aryl, heteroaryl, —S(O)₂NR¹³R¹⁴ and —SO₂R²⁰ (wherein R²⁰ is alkyl, aryl,aralkyl, heteroaryl and heteroaralkyl);

R³ is selected from the group consisting of hydrogen, halogen, alkyl,trihalomethyl, hydroxy, alkoxy, —(CO)R¹⁵, —NR¹³R¹⁴, aryl, heteroaryl,—NR¹³S(O)₂R¹⁴, —S(O)₂NR¹³R¹⁴, —NR¹³C(O)R¹⁴, —NR¹³C(O)OR¹⁴ and —SO₂R²⁰(wherein R²⁰ is alkyl, aryl, aralkyl, heteroaryl and heteroaralkyl);

R⁴ is selected from the group consisting of hydrogen, halogen, alkyl,hydroxy, alkoxy and —NR¹³R¹⁴;

R⁵ is selected from the group consisting of hydrogen, alkyl and—C(O)R¹⁰;

R⁶ is selected from the group consisting of hydrogen, alkyl and—C(O)R¹⁰;

R⁷ is selected from the group consisting of hydrogen, alkyl, aryl,heteroaryl, —C(O)R¹⁷ and —C(O)R¹⁹; or

R⁶ and R⁷ may combine to form a group selected from the group consistingof —(CH₂)₄ —, —(CH₂)₅— and —(CH₂)₆—; with the proviso that at least oneof R⁵, R⁶ or R⁷ must be —C(O)R¹⁰;

R⁸ and R⁹ are independently selected from the group consisting ofhydrogen, alkyl and aryl;

R¹⁰ is selected from the group consisting of hydroxy, alkoxy, aryloxy.—N(R¹¹) (CH₂)_(n)R¹², and —NR¹³R¹⁴;

R¹¹ is selected from the group consisting of hydrogen and alkyl;

R¹² is selected from the group consisting of —NR¹³R¹⁴, hydroxy,—C(O)R¹⁵, aryl, heteroaryl, —N⁴(O⁻)R¹³R¹⁴, —N(OH)R¹³, and —NHC(O)R^(a)(wherein R^(a) is unsuhstituted alkyl, haloalkyl, or aralkyl);

R¹³ and R¹⁴ are independently selected from the group consisting ofhydrogen, alkyl, cyanoalkyl, cyvcloalkyl, aryl and heteroaryl; or

R¹³ and R¹⁴ may combine to form a heterocyclo group;

R¹⁵ is selected from the group consisting of hydrogen, hydroxy, andaryloxy;

R¹⁶ is selected from the group consisting of hydroxy, —C(O)R¹⁵, —NR¹³R¹⁴and —C(O)NR¹³R¹⁴;

R¹⁷ is selected from the group consisting of alkyl, cycloalkyl, aryl andheteroaryl;

R²⁰ is alkyl, aryl, aralkyl or heteroaryl; and

n and r are independently 1, 2, 3, or 4;

or a pharmaceutically acceptable salt thereof.

In certain embodiments, the compound of formula 1 has the formula;

The following definitions are used herein:

“Alkyl” refers to a saturated aliphatic hydrocarbon radical includingstraight chain and branched chain groups of 1 to 20 carbon atoms(whenever a numerical range; e.g. “1-20”, is stated herein, it meansthat the group, in this case the alkyl group, may contain 1 carbon atom,2 carbon atoms, 3 carbon atoms, etc. up to and including 20 carbonatoms). Alkyl groups containing from 1 to 4 carbon atoms are referred toas lower alkyl groups. When the lower alkyl groups lack substituents,they are referred to as unsubstituted lower alkyl groups. Morepreferably, an alkyl group is a medium size alkyl having 1 to 10 carbonatoms e.g., methyl, ethyl, propyl, 2-propyl, n-butyl, iso-butyl,tert-butyl, and pentyl. Most preferably, it is a lower alkyl having 1 to4 carbon atoms e.g., methyl, ethyl, propyl, 2-propyl, n-butyl,iso-butyl, or tert-butyl. The alkyl group may be substituted orunsubstituted. When substituted, the substituent group(s) is preferablyone or more, more preferably one to three, even more preferably one ortwo substituent(s) independently selected from the group consisting ofhalo, hydroxy, unsubstituted lower alkoxy, aryl optionally substitutedwith one or more groups, preferably one, two or three groups which areindependently of each other halo, hydroxy, unsubstituted lower alkyl orunsubstituted lower alkoxy groups, aryloxy optionally substituted withone or more groups, preferably one, two or three groups which areindependently of each other halo, hydroxy, unsubstituted lower alkyl orunsubstituted lower alkoxy groups, 6-member heteroaryl having from 1 to3 nitrogen atoms in the ring, the carbons in the ring being optionallysubstituted with one or more groups, preferably one, two or three groupswhich are independently of each other halo, hydroxy, unsubstituted loweralkyl or unsubstituted lower alkoxy groups, 5-member heteroaryl havingfrom 1 to 3 heteroatoms selected from the group consisting of nitrogen,oxygen and sulfur, the carbon and the nitrogen atoms in the group beingoptionally substituted with one or more groups, preferably one, two orthree groups which are independently of each other halo, hydroxy,unsubstituted lower alkyl or unsubstituted lower alkoxy groups, 5- or6-member heteroalicyclic group having from 1 to 3 heteroatoms selectedfrom the group consisting of nitrogen, oxygen and sulfur, the carbon andnitrogen (if present) atoms in the group being optionally substitutedwith one or more groups, preferably one, two or three groups which areindependently of each other halo, hydroxy, unsubstiutted lower alkyl orunsubstituted lower alkoxy groups, mercapto, (unsubstituted loweralkyl)thio, aryithio optionally substituted with one or more groups,preferably one, two or three groups which are independently of eachother halo, hydroxy, unsubstituted lower alkyl or unsubstituted loweralkoxy groups, cyano, acyl, thioacyl, O-carbamyl, N-carbamyl,O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, nitro, N-surfonamido,S-sulfonamido, R¹⁸S(O)—, R¹⁸S(O)₂—, C(O)OR¹⁸, R¹⁸c(O)O—, and —NR¹⁸R¹⁹,wherein R¹⁸ and R¹⁹ are independently selected from the group consistingof hydrogen, unsubstituted lower alkyl, trihalomethyl, unsubstituted(C₃-C₆)cycloalkyl. unsubstituted lower alkenyl, unsubstituted loweralkynyl and aryl optionally substituted with one or more, groups,preferably one, two or three groups which are independently of eachother halo, hydroxy, unsubstituted lower alkyl or unsubstituted loweralkoxy groups.

Preferably, the alkyl group is substituted with one or two substituentsindependently selected from the group consisting of hydroxy, 5- or6-member heteroalicyclic group having from 1 to 3 heteroatoms selectedfrom the group consisting of nitrogen, oxygen and sulfur, the carbon andnitrogen (if present) atoms m the group being optionally substitutedwith one or more groups, preferably one, two or three groups which areindependently of each other halo, hydroxy, unsubstituted lower alkyl orunsubstituted lower alkoxy groups, 5-member heteroaryl having from 1 to3 heteroatoms selected from the group consisting of nitrogen, oxygen andsulfur, the carbon and the nitrogen atoms in the group being optionallysubstituted with one or more groups, preferably one, two or three groupswhich are independently of each other halo, hydroxy, unsubstituted loweralkyl or unsubstituted lower alkoxy groups, 6-member heteroaryl havingfrom 1 to 3 nitrogen atoms in the ring, the carbons in the ring beingoptionally substituted with one or more groups, preferably one, two orthree groups which are independently of each other halo, hydroxy,unsubstituted lower alkyl or unsubstituted lower alkoxy groups, or—NR¹⁸R¹⁹, wherein R¹⁸ and R¹⁹ are independently selected from the groupconsisting 0f hydrogen, unsubstituted lower alkyl. Even more preferablythe alkyl group is substituted with one or two substituents which areindependently of each other hydroxy, dimethylamino, ethylamino,diethylamino, dipropylamino, pyrrolidino, piperidine, morpholino,piperazino, 4-lower alkylpiperazino, phenyl, imidazolyl, pyridinyl,pyridazinyl, pyrimidinyl, oxazolyl, and triazinyl.

“Cycloalkyl” refers to a 3 to 8 member all-carbon monocyclic ring, anall-carbon 5-member/6-member or 6-member/6-member fused bicyclic ring ora multicyclic fused ring (a “fused” ring system means that each ring inthe system shares an adjacent pair of carbon atoms with each other ringin the system) group wherein one or more of the rings may contain one ormore double bonds but none of the rings has a completely conjugatedpi-electron system. Examples of cycloalkyl groups are cyclopropane,cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclahexadiene,adamantine, cycloheptane, and cyclaheptatriene. A cycloalkyl group maybe substituted or unsubstituted. When substituted, the substituentgroup(s) is preferably one or more, more preferably one or twosubstituents, independently selected from the group consisting ofunsubstituted lower alkyl, trihaloalkyl, halo, hydroxy, unsubstitutedlower alkoxy, aryl optionally substituted with one or more, preferablyone or two groups independently of each other halo, hydroxy,unsubstituted lower alkyl or unsubstituted lower alkoxy groups, aryloxyoptionally substituted with one or more, preferably one or two groupsindependently of each other halo, hydroxy, unsubstituted lower alkyl orunsubstituted lower alkoxy groups, 6-member heteroaryl having from 1 to3 nitrogen atoms in the ring, the carbons in the ring being optionallysubstituted with one or more, preferably one or two groups independentlyof each other halo, hydroxy, unsubstituted lower alkyl or unsubstitutedlower alkoxy groups, 5-member heteroaryl having from 1 to 3 heteroatomsselected from the group consisting of nitrogen, oxygen and sulfur, thecarbon and nitrogen atoms of the group being optionally substituted withone or more, preferably one or two groups independently of each otherhalo, hydroxy, unsubstituted lower alkyl or unsubstituted lower alkoxygroups, 5- or 6-member heteroalicyclic group haying from 1 to 3heteroatoms selected from the group consisting of nitrogen, oxygen andsulfur, the carbon and nitogen (if present) atoms in the group beingoptionally substituted with one or more, preferably one or two groupsindependently of each other halo, hydroxy, unseahstituted. lower alkylor unsubstituted lower alkoxy groups, mercapto, (unsubstituted loweralkyl)thio, aryl thio optionally substituted with one or more,preferably one or two groups independently of each other halo, hydroxy,unsubstituted lower alkyl or unsubstituted lower aikoxy groups, cyano,acyl, thioacyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,C-amido, N-amido, nitro, N-sulfonamido, S-sulfonamido, R¹⁸S(O)—,R¹⁸S(O)₂—, —C(O)OR¹⁸, R¹⁸C(O)O—, and NR¹⁸R¹⁹ are as defined above.

“Alkenyl” refers to a lower alkyl group, as defined herein, consistingof at least two carbon atoms and at least one carbon-carbon double bond.Representative examples include, but are not limited to, ethenyl,1-propenyl, 2-propenyl, and 1-, 2-, or 3-butenyl.

“Alkenyl” refers to a lower alkyl group, as defined herein, consistingof at least two carbon atoms and at least one carbon-carbon triple bond.Representative examples include, but are not limited to, ethynyl,1propynyl, 2-propynyl, and 1-, 2-, or 3-butynyl.

“Aryl” refers to an all-carbon monocyclic or fused-ring polycyclic ringswhich share adjacent pairs of carbon atoms) groups of 1 to 12 carbonatoms having a completely conjugated pi-electron system. Examples,without limitation, of aryl groups are phenyl, naphthalenyl andanthracenyl. The aryl group may be substituted or unsubstituted. Whensubstituted, the substituted group(s) is preferably one or more, morepreferably one, two or three, even more preferably one or two,independently selected from the group consisting of unsubstituted loweralkyl, trihaloalkyl, halo, hydrox, unsubstituted lower alkoxy,mercapto,(unsubstituted lower alkyl)thio, cyano, acyl, thioacyl,O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido,N-amido, nitro, N-sulfonamido, S-sulfonamido, R¹⁸S(O)—, R¹⁸S(O)₂—;—C(O)OR¹⁸, R¹⁸C(O)O—, and NR¹⁸R¹⁹, with R¹⁸and R¹⁹ as defined above.Preferably, the aryl group is optionally substituted with one or twosubstituents independently selected from halo, unsubstituted loweralkyl, trihaloalkyl, hydroxy, mercapto, cyano, N-amido, mono ordialkylamino, carboxy, or N-sulforiamido.

“Heteroaryl” refers to a monocyclic or fused ring (i.e., rings whichshare an adjacent pair of atoms) group of 5 to 12 ring atoms containingone, two, or three ring heteroatoms selected from N, O, or S, theremaining ring atoms being C, and, in addition, having a completelyconjugated pi-electron system. Examples, without limitation, ofunsubstituted heteroaryl groups are pyrrole, furan, thiophene,imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline,isoquinoline, purine and carbazole. The heteroaryl group may besubstituted or unsubstituted. When substituted, the substituted group(s)is preferably one or more, more preferably one, two, or three, even morepreferably one or two, independently selected from the group consistingof unsubstituted lower alkyl, trihaloalkyl, halo, hydroxy, unsubstitutedlower alkoxy, mercapto, (unsubstituted lower alkyl)thio, cyano, acyl,thioacyl, o-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,C-amido, N-amido, nitro, N-sulfonamido, S-sulfonamido, R¹⁸S(O)—,R¹⁸O)₂—, —C(O)OR¹⁸, R¹⁸C(O)O—, and NR¹⁸R¹⁹, with R¹⁸ and R¹⁹ as definedabove. Preferably, the heteroaryl group is optionally substituted withone or two substituents independently selected from halo, unsubstitutedlower alkyl, trihaloalkyl, hydroxy, mercapto, cyano, N-amido, mono ordialkylamino, carboxy. or N-sulfonamido.

“Heteroalicyclic” refers to a monocyclic or fused ring group having inthe ring(s) of 5 to 9 ring atoms in which one or two ring atoms areheteroatoms selected from N, O, or S(O)_(n) (where n is an integer from0 to 2), the remaining ring atoms being C. The rings may also have oneor more double bonds. However, the rings do not have a completelyconjugated pi-electron system. Examples, without limitation, ofunsubstituted heteroalicyclic groups are pyrrolidino, piperidino,piperazino, morpholino, thiomorpholino, and homopiperazino. Theheteroalicyclic ring may be substituted or unsubstituted. Whensubstituted, the substituted group(s) is preferably one or more, morepreferably one, two or three, even more preferably one or two,independently selected from the group consisting of unsubstituted loweralkyl, trihaloalkyl, halo, hydroxy, unsubstituted lower alkoxy,mercapto, (unsubstituted lower alkyl)thio, cyano, acyl, thioacyl,O-carbamyl, N-carbamyl, O-thiocarbarnyl, N-thiocarbamyl, C-amido,N-amido, nitro, N-sulfonamido, S-sulfonamido, R¹⁸S(O)—R¹⁸S(O)₂—,—C(O)OR¹⁸, R¹⁸C(O)O—, and —NR¹⁸R¹⁹, with R¹⁸ and R¹⁹ as defined above.Preferably, the heteroalicyclic group is optionally substituted with oneor two substituents independently selected from halo, unsubstitutedlower alkyl, trihaloalkyl, hydroxy, mercapto, cyano, N-amido, mono ordialkylamino, carboxy, or N-sulfonamido.

Preferably, the heteroalicyclic group is optionally substituted with oneor two substituents independently selected from halo, unsubstitutedlower alkyl, trihaloalkyl, hydroxy, mercapto, cyano, N-amido, mono ordialkylamino. carboxy, or N-sulfonamido.

“Heterocycle” means a saturated cyclic radical of 3 to 8 ring atoms inwhich one or two ring atoms are heteroatoms selected from N, O, orS(O)_(n) (where n is an integer from 0 to 2), the remaining ring atomsbeing C, where one or two C atoms may optionally be replaced by acarbonyl group. The heterocyclyl ring may be optionally substitutedindependently with one, two, or three substituents selected fromoptionally substituted lower alkyl (substituted with 1 or 2 substituentsindependently selected from carboxy or ester), haloalkyl, cyanoalkyl,halo, nitro, cyano, hydroxy, alkoxy, amino, monoalkylamino,dialkylamino, aralkyl. heteroaralkyl, —COR (where R is alkyl) or —COORwhere R is (hydrogen or alkyl), More specifically the term heterocyclylincludes, but is not limited to, tetrahydropyranyl,2,2-dimethyl-1,3-dioxolane, piperidines, piperazino, N-methylpyrrolidin3-yl, 3-pyrrolidine, morpholino, thiomorpholino, thiomorpholino-1-oxide,thiomorpholino 1,1-dioxide, 4-ethyloxycarbonylpiperazino,3-oxopiperazino, 2-imidazolidone, 2-pyrrolidinone, 2-oxohoniopiperazino,tetrahydropyrimidin-2-one, and the derivatives thereof. Preferably, theheterocycle group is optionally substituted with one or two substituentsindependently selected from halo, unsubstituted lower alkyl, lower alkylsubstituted with carboxy, ester, hydroxy, mono or dialkylamino.

“Hydroxy” refers to an —OH group.

“Alkoxy” refers to both an—O-(unsubstituted alkyl) and an—O-(unsubstituted cycloalkyli group. Representative examples include,but are not limited to, e.g., methoxy, ethoxy, propoxy, butoxy,cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, and cyclohexyloxy.

“Aryloxy” refers to both an O-aryl and an O-heteroaryl group, as definedherein. Representative examples include, but are not limited to,phenoxy, pyridinyloxy, furanyloxy, thienyloxy, pyrimidinyloxy,pyrazinyloxy, and derivatives thereof.

“Mercapto” refers to an —SH group.

“Alkyithio” refers to both an —S-(unsubstituted alkyl) and an—S-(unsubstituted cycloalkyl) group. Representative examples include,but are not limited to, e.g., methylthio, ethylthio, propylthio,butylthio, cyclopropylthio, cyclobutylthio, cvclopentylthio, andcyclohexylthio.

“Arylthio” refers to both an —S-aryl and an —S-heteroarvl group, asdefined herein. Representative examples include, but are not limited to,phenyithio, pyridinylthio, furanylthio, thientylthio, pyrimidinylthio,and derivatives thereof.

“Acyl” refers to a —C(O)—R″ group, where R″ is selected from the groupconsisting of hydrogen, unsubstituted lower alkyl, trihalomethyl,unsubstituted cycloalkyl, aryl optionally substituted with one or more,preferably one, two, or three substituents selected from the groupconsisting of unsubstituted lower alkyl, trihalomethyl, unsubstitutedlower alkoxy, halo and —NR¹⁸R¹⁹ groups, heteroaryl (bonded through aring carbon) optionally substituted with one or more, preferably one,two, or three substitutents selected from the group consisting ofunsubstituted lower alkyl, trihaloalkyl, unsubstituted lower alkoxy,halo and —NR¹⁸R¹⁹ groups and heteroalicyclic (bonded through a ringcarbon) optionally substituted with one or more, preferably one, two, orthree substituents selected from the group consisting of unsubstitutedlower alkyl, trihaloalkyl, unsubstituted lower alkoxy, halo and —NR¹⁸R¹⁹groups. Representative acy groups include, but are not limited to,acetyl, trifluoroacetyl, and benzoyl.

“Aldehyde” refers to an acyl group in which R″ is hydrogen.

“Thioacyl” refers to a —C(S)—R″ group, with R″ as defined herein.

“Ester” refers to a —C(O)O—R″ group with R″ as defined herein exceptthat R″ cannot be hydrogen.

“Acetyl” group refers to a —C(O)CH₃ group.

“Halo” group refers to fluorine, chlorine, bromine or iodine, preferablyfluorine or chlorine.

“Trihalomethyl” group refers to a —CX₃ group wherein X is a halo groupas defined herein.

“Trihalomethanesulfonyl” group refers to a X₃CS(═O)₂— groups with X asdefined above.

“Cyano” refers to a —C≡N group.

“Methylenedioxy” refers to a —OCH₂O— group where the two oxygen atomsare bonded to adjacent carbon atoms.

“Ethylenedioxy” group refers to a —OCH₂CH₂O— where the two oxygen atomsare bonded to adjacent carbon atoms.

“S-sulfonamido” refers to a —S(O)₂NR¹⁸R¹⁹ group, with R¹⁸ and R¹⁹ asdefined herein. “N-sulfonamido” refers to a —NR¹⁸S(O)₂R¹⁹ group, withR¹⁸ and R¹⁹ as defined herein.

“'O-carbamyl” group refers to a —OC(O)NR¹⁸R¹⁹ group with R¹⁸ and R¹⁹ asdefined herein. “N-carbamyl” refers to an R¹⁸OC(O)NR¹⁹— group, with R¹⁸and R¹⁹ as defined herein.

“O-thiocarbamyl” refers to a —OC(S)NR¹⁸R¹⁹ group with R¹⁸ and R¹⁹ asdefined herein. “N-thiocarbamyl” refers to a R¹⁸OC(S)NR¹⁹— group, withR¹⁸ and R¹⁹ as defined herein.

“Amino” refers to an —NR¹⁸R¹⁹group, wherein R¹⁸ and R¹⁹ are bothhydrogen.

“C-amido” refers to a —C(O)NR¹⁸R¹⁹ group with R¹⁸ and R¹⁹ as definedherein. “N-amide” refers to a R¹⁸C(O)NR¹⁹ group, with R¹⁸ and R¹⁹ asdefined herein.

“Nitro” refers to a —NO₂ group.

“Haloalkyl” means an unsubstituted aikyl, preferably unsubstituted loweralkyl as defined above that is substituted with one or more same ordifferent halo atoms, e.g., —CH₂Cl, —CF₃, —CH₂CF₃, and —CH₂CCl₃.

“Aralkyl” means unsubstituted alkyl, preferably unsubstituted loweralkyl as defined above which is substituted with an aryl group asdefined above, e.g., —CH₂phenyl, —(CH₂)₂phenyl, —(CH₂)₃phenyl,CH₃CH(CH₃)CH₂phenyl, and derivatives thereof.

“Heteroaralkyl” group means unsubstituted alkyl, preferablyunsubstituted lower alkyl as defined above, e.g., —CH₂phenyl,—(CH₂)₂phenyl, —(CH₂)₃phenyl, CH₃CH(CH₃)CH₂phenyl, and derivativesthereof.

“Dialkylamino” means a radical —NRR where each R is independently anunsubstitued alkyl or unsubstituted cycloalkyl group as defined above,e.g., dimethylamino, diethylamino, (1-methylethyl)-ethylamino,cyclohexylmethylamino, and cyclopentylmethylamino.

“Cyanoalkyl” means unsubstituted alkyl, preferably unsubstituted loweralkyl as defined above, which is substituted with 1 or 2 cyano groups.

“Optional” or “optionally” means that the subsequently described eventor circumstance may but need not occur, and that the descriptionincludes instances where the event or circumstance occurs and instancesin which it does not. For example, “heterocycle group optionallysubstituted with an alkyl group” means that the alkyl may but need notbe present, and the description includes situations where theheterocycle group is substituted with an alkyl group and situationswhere the heterocyclo group is not substituted with the alkyl group.

B. Encapsulating Polymers

Controlled release dosage formulations for the delivery of one or moredrugs in a polymeric vehicle are described herein. The polymeric matrixcan be formed from non-biodegradable or biodegradable polymers; however,the polymer matrix is preferably biodegradable. The polymeric matrix canbe formed into implants (e.g., rods, disks, wafers, etc.),microparticles, nanoparticles, or combinations thereof for delivery.Upon administration, the sunitinib is released over an extended periodof time, either upon degradation of the polymer matrix, diffusion of theone or more inhibitors out of the polymer matrix, or a combinationthereof. The drug can be dispersed or encapsulated into the polymer orcovalently bound to the polymer used to form the matrix. The degradationprofile of the one or more polymers may be selected to influence therelease rate of the active agent in vivo.

The polymers may be hydrophobic, hydrophilic, conjugates of hydrophilicand hydrophobic polymers (i.e., amphiphilic polymers), blockco-polymers, and blends thereof.

Examples of suitable hydrophobic polymers include, but are not limitedto, polyhydroxyesters such as polylactic acid, polyglycolic acid, orcopolymers thereof, polycaprolactone, polyanhydrides such as polysebacicanhydride, and copolymers of any of the above.

The one or more hydrophilic polymers can be any hydrophilic,biocompatible, non-toxic polymer or copolymer. In certain embodiments,the one or more hydrophilic polymers contain a poly(alkylene glycol),such as polyethylene glycol (PEG). In particular embodiments, the one ormore hydrophilic polymers are linear PEG chains.

Representative synthetic polymers include poly(hydroxy acids) such aspoly(lactic acid), poly(glycolic acid), and poly(lactic acid-co-glycolicacid), poly(lactide), poly(glycolide), poly(lactide-co-glycolide),polyanhydrides, polyorthoesters, polyamides, polycarbonates,polyalkylenes such as polyethylene and polypropylene, polyalkyleneglycols such as poly(ethylene glycol), polyalkylene oxides such aspoly(ethylene oxide), polyalkylene terephthalates such as poly(ethyleneterephthalate), polyvinyl alcohols, polyvinyl ethers, polyvinyl esters,polyvinyl halides such as poly(vinyl chloride), polyvinylpyrrolidone,polysiloxanes, poly(vinyl alcohols), poly(vinyl acetate), polystyrene,polyurethanes and co-polymers thereof, celluloses such as alkylcellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters,nitro celluloses, methyl cellulose, ethyl cellulose, hydroxypropylcellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methylcellulose, cellulose acetate, cellulose propionate, cellulose acetatebutyrate, cellulose acetate phthalate, carboxylethyl cellulose,cellulose triacetate, and cellulose sulphate sodium salt (jointlyreferred to herein as “celluloses”), polymers of acrylic acid,methacrylic acid or copolymers or derivatives thereof including esters,poly(methyl methacrylate), poly(ethyl methacrylate),poly(butylmethacrylate), poly(isobutyl methacrylate),poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecylacrylate) (jointly referred to herein as “polyacrylic acids”),poly(butyric acid), poly(valeric acid), andpoly(lactide-co-caprolactone), copolymers and blends thereof. As usedherein, “derivatives” include polymers having substitutions, additionsof chemical groups, for example, alkyl, alkylene, hydroxylations,oxidations, and other modifications routinely made by those skilled inthe art.

Examples of preferred natural polymers include proteins such as albuminand prolamines, for example, zein, and polysaccharides such as alginate,cellulose and polyhydroxyalkanoates, for example, polyhydroxybutyrate.

Examples of preferred non-biodegradable polymers include ethylene vinylacetate, poly(meth)acrylic acid, polyamides, copolymers and mixturesthereof.

C. Alkalizing Agents

It was discovered that the loading of sunitinib can be increased byincreasing the alkalinity of the sunitinib in solution duringencapsulation. This can be achieved by selection of the solvent, addingalkalilizing agents to the solvent, or including alkaline drugs with thesunitini. Examples of compounds that can be added for this purposeinclude solvents or solvent additives such as DMA, DMTA, TEA, aniline,ammonium, and sodium hydroxide, drugs such as Vitamin B4, caffeine,alkaloids, nicotine, the analgesic morphine, the antibacterialberberine, the anticancer compound vincristine, the antihypertensionagent reserpine, the cholinomimetic galantamine, the anticholinergicagent atropine, the vasodilator vincamine, the antiarrhythmia compoundquinidine, the antiasthma therapeutic ephedrine, and the antimalarialdrug quinine.

III. Methods of Forming Microparticles, Nanoparticles and Implants A.Micro and Nanoparticle Formation

Microparticle and nanoparticles can be formed using any suitable methodfor the formation of polymer micro- or nanoparticles known in the art.The method employed for particle formation will depend on a variety offactors, including the characteristics of the polymers present in thedrug or polymer matrix, as well as the desired particle size and sizedistribution. The type of drug(s) being incorporated in the particlesmay also be a factor as some Drugs are unstable in the presence ofcertain solvents, in certain temperature ranges, and/or in certain pHranges.

Particles having an average particle size of between 10 nm and 1000microns are useful in the compositions described herein. In preferredembodiments, the particles have an average particle size of between 10nm and 100 microns, more preferably between about 100 nm and about 50microns, more preferably between about 200 nm and about 50 microns. Theparticles can have any shape but are generally spherical in shape.

Preferably, particles formed from one or more drugs contain significantamounts of a hydrophilic polymer, such as PEG, on their surface. Incircumstances where a monodisperse population of particles is desired,the particles may be formed using a method which produces a monodispersepopulation of nanoparticles. Alternatively, methods producingpolydisperse nanoparticle distributions can be used, and the particlescan be separated using methods known in the art, such as sieving,following particle formation to provide a population of particles havingthe desired average particle size and particle size distribution.

Common techniques for preparing microparticles and nanoparticlesinclude, but are not limited to, solvent evaporation, hot melt particleformation, solvent removal, spray drying, phase inversion, coacervation,and low temperature casting. Suitable methods of particle formulationare briefly described below. Pharmaceutically acceptable excipients,including pH modifying agents, disintegrants, preservatives, andantioxidants, can optionally be incorporated into the particles duringparticle formation.

1. Solvent Evaporation

In this method, the drug (or polymer matrix and one or more Drugs) isdissolved in a volatile organic solvent, such as methylene chloride. Theorganic solution containing the drug is then suspended in an aqueoussolution that contains a surface active agent such as poly(vinylalcohol). The resulting emulsion is stirred until most of the organicsolvent evaporated, leaving solid nanoparticles. The resultingnanoparticles are washed with water and dried overnight in a lyophilizerNanoparticles with different sizes and morphologies can be obtained bythis method.

Drugs which contain labile polymers, such as certain polyanhydrides, maydegrade during the fabrication process due to the presence of water. Forthese polymers, the following two methods, which are performed incompletely anhydrous organic solvents, can be used.

2. Solvent Removal

Solvent removal can also be used to prepare particles from drugs thatare hydrolytically unstable. In this method, the drug (or polymer matrixand one or more Drugs) is dispersed or dissolved in a volatile organicsolvent such as methylene chloride. This mixture is then suspended bystirring in an organic oil (such as silicon oil) to form an emulsion.Solid particles form from the emulsion, which can subsequently beisolated from the supernatant. The external morphology of spheresproduced with this technique is highly dependent on the identity of thedrug.

3. Spray Drying

In this method, the drug (or polymer matrix and one or more Drugs) isdissolved in an organic solvent such as methylene chloride. The solutionis pumped through a micronizing nozzle driven by a flow of compressedgas, and the resulting aerosol is suspended in a heated cyclone of air,allowing the solvent to evaporate from the microdroplets, formingparticles. Particles ranging between 0.1-10 microns can be obtainedusing this method.

4. Phase Inversion

Particles can be formed from drugs using a phase inversion method. Inthis method, the drug (or polymer matrix and one or more Drugs) isdissolved in a “good” solvent, and the solution is poured into a strongnon solvent for the drug to spontaneously produce, under favorableconditions, microparticles or nanoparticles. The method can be used toproduce nanoparticles in a wide range of sizes, including, for example,about 100 nanometers to about 10 microns, typically possessing a narrowparticle size distribution.

5. Coacervation

Techniques for particle formation using coacervation are known in theart, for example, in GB-B-929 406; GB-B-929 40 1; and U.S. Pat. Nos.3,266,987, 4,794,000, and 4,460,563. Coacervation involves theseparation of a drug (or polymer matrix and one or more Drugs)solutioninto two immiscible liquid phases. One phase is a dense coacervatephase, which contains a high concentration of the drug, while the secondphase contains a low concentration of the drug. Within the densecoacervate phase, the drug forms nanoscale or microscale droplets, whichharden into particles. Coacervation may be induced by a temperaturechange, addition of a non-solvent or addition of a micro-salt (simplecoacervation), or by the addition of another polymer thereby forming aninterpolymer complex (complex coacervation).

6. Low Temperature Casting

Methods for very low temperature casting of controlled releasemicrospheres are described in U.S. Pat. No. 5,019,400 to Gombotz et al.In this method, the drug (or polymer matrix and sunitinib) is dissolvedin a solvent. The mixture is then atomized into a vessel containing aliquid non-solvent at a temperature below the freezing point of the drugsolution which freezes the drug droplets. As the droplets andnon-solvent for the drug are warmed, the solvent in the droplets thawsand is extracted into the non-solvent, hardening the microspheres.

D. Implants

Implants can be formed which encapsulate and/or have dispersed thereinthe drug. In preferred embodiments, the implants are intraocularimplants. Suitable implants include, but are not limited to, rods,discs, and wafers. The matrix can be formed of any of thenon-biodegradable or biodegradable polymers described above, althoughbiodegradable polymers are preferred. The composition of the polymermatrix is selected based on the time required for in vivo stability,i.e. that time required for distribution to the site where delivery isdesired, and the time desired for delivery. The implants may be of anygeometry such as fibers, sheets, films, microspheres, spheres, circulardiscs, rods, or plaques. Implant size is determined by factors such astoleration for the implant, location of the implant, size limitations inview of the proposed method of implant insertion, ease of handling, etc.

Where sheets or films are employed, the sheets or films will be in therange of at least about 0.5 mm×0.5 mm, usually about 3 to 10 mm×5 to 10mm with a thickness of about 0.1 to 1.0 mm for ease of handling. Wherefibers are employed, the fiber diameter will generally be in the rangeof about 0.05 to 3 mm and the fiber length will generally be in therange of about 0.5 to 10 mm

The size and shape of the implant can also be used to control the rateof release, period of treatment, and drug concentration at the site ofimplantation. Larger implants will deliver a proportionately largerdose, but depending on the surface to mass ratio, may have a slowerrelease rate. The particular size and geometry of the implant are chosento suit the site of implantation.

Intraocular implants may be spherical or non-spherical in shape. Forspherical-shaped implants, the implant may have a largest dimension(e.g., diameter) between about 5 um and about 2 mm, or between about 10um and about 1 mm for administration with a needle, greater than 1 mm,or greater than 2 mm, such as 3 mm or up to 10 mm, for administration bysurgical implantation. If the implant is non-spherical, the implant mayhave the largest dimension or smallest dimension be from about 5 um andabout 2 mm, or between about 10 um and about 1 mm for administrationwith a needle, greater than 1 mm, or greater than 2 mm, such as 3 mm orup to 10 mm, for administration by surgical implantation.

The vitreous chamber in humans is able to accommodate relatively largeimplants of varying geometries, having lengths of, for example, 1 to 10mm The implant may be a cylindrical pellet (e.g., rod) with dimensionsof about 2 mm×0.75 mm diameter. The implant may be a cylindrical pelletwith a length of about 7 mm to about 10 mm, and a diameter of about 0.75mm to about 1.5 mm In certain embodiments, the implant is in the form ofan extruded filament with a diameter of about 0.5 mm, a length of about6 mm, and a weight of approximately 1 mg. In some embodiments, thedimension are, or are similar to, implants already approved forintraocular injection via needle: diameter of 460 microns and a lengthof 6 mm and diameter of 370 microns and length of 3.5 mm.

Intraocular implants may also be designed to be least somewhat flexibleso as to facilitate both insertion of the implant in the eye, such as inthe vitreous, and subsequent accommodation of the implant. The totalweight of the implant is usually about 250 to 5000 μg, more preferablyabout 500-1000 μg. In certain embodiments, the intraocular implant has amass of about 500 μg, 750 μg, or 1000 μg.

2. Methods of Manufacture

Implants can be manufactured using any suitable technique known in theart. Examples of suitable techniques for the preparation of implantsinclude solvent evaporation methods, phase separation methods,interfacial methods, molding methods, injection molding methods,extrusion methods, coextrusion methods, carver press method, die cuttingmethods, heat compression, and combinations thereof. Suitable methodsfor the manufacture of implants can be selected in view of many factorsincluding the properties of the polymer/polymers present in the implant,the properties of the one or more drugs present in the implant, and thedesired shape and size of the implant. Suitable methods for thepreparation of implants are described, for example, in U.S. Pat. No.4,997,652 and U.S. Patent Application Publication No. US 2010/0124565.

In certain cases, extrusion methods may be used to avoid the need forsolvents during implant manufacture. When using extrusion methods, thepolymer/polymers and Drug are chosen so as to be stable at thetemperatures required for manufacturing, usually at least about 85° C.However, depending on the nature of the polymeric components and the oneor more Drugs, extrusion methods can employ temperatures of about 25° C.to about 150° C., more preferably about 65° C. to about 130° C. Implantsmay be coextruded in order to provide a coating covering all or part ofthe surface of the implant. Such coatings may be erodible ornon-erodible, and may be impermeable, semi-permeable, or permeable tothe Drug, water, or combinations thereof. Such coatings can be used tofurther control release of the Drug from the implant.

Compression methods may be used to make the implants. Compressionmethods frequently yield implants with faster release rates thanextrusion methods. Compression methods may employ pressures of about50-150 psi, more preferably about 70-80 psi, even more preferably about76 psi, and use temperatures of about 0° C. to about 115° C., morepreferably about 25° C.

IV. Pharmaceutical Formulations A. Pharmaceutical Excipients

Pharmaceutical formulations contain sunitinib in combination with one ormore pharmaceutically acceptable excipients. Representative excipientsinclude solvents, diluents, pH modifying agents, preservatives,antioxidants, suspending agents, wetting agents, viscosity modifiers,tonicity agents, stabilizing agents, and combinations thereof. Suitablepharmaceutically acceptable excipients are preferably selected frommaterials which are generally recognized as safe (GRAS), and may beadministered to an individual without causing undesirable biologicalside effects or unwanted interactions.

In some cases, the pharmaceutical formulation contains only one type ofconjugate or polymeric particles for the controlled release of Drugs(e.g., a formulation containing drug particles wherein the drugparticles incorporated into the pharmaceutical formulation have the samecomposition). In other embodiments, the pharmaceutical formulationcontains two or more different type of conjugates or polymeric particlesfor the controlled release of Drugs (e.g., the pharmaceuticalformulation contains two or more populations of drug particles, whereinthe populations of drug particles have different chemical compositions,different average particle sizes, and/or different particle sizedistributions).

Particles formed from the drugs will preferably be formulated as asolution or suspension for injection to the eye or into a tissue such asa tumor.

Pharmaceutical formulations for ocular administration are preferably inthe form of a sterile aqueous solution or suspension of particles formedfrom sunitinib. Acceptable solvents include, for example, water,Ringer's solution, phosphate buffered saline (PBS), and isotonic sodiumchloride solution. The formulation may also be a sterile solution,suspension, or emulsion in a nontoxic, parenterally acceptable diluentor solvent such as 1,3-butanediol.

In some instances, the formulation is distributed or packaged in aliquid form. Alternatively, formulations for ocular administration canbe packed as a solid, obtained, for example by lyophilization of asuitable liquid formulation. The solid can be reconstituted with anappropriate carrier or diluent prior to administration.

Solutions, suspensions, or emulsions for ocular administration may bebuffered with an effective amount of buffer necessary to maintain a pHsuitable for ocular administration. Suitable buffers are well known bythose skilled in the art and some examples of useful buffers areacetate, borate, carbonate, citrate, and phosphate buffers.

Solutions, suspensions, or emulsions for ocular administration may alsocontain one or more tonicity agents to adjust the isotonic range of theformulation. Suitable tonicity agents are well known in the art and someexamples include glycerin, mannitol, sorbitol, sodium chloride, andother electrolytes.

Solutions, suspensions, or emulsions for ocular administration may alsocontain one or more preservatives to prevent bacterial contamination ofthe ophthalmic preparations. Suitable preservatives are known in theart, and include polyhexamethylenebiguanidine (PHMB), benzalkoniumchloride (BAK), stabilized oxychloro complexes (otherwise known asPurite®), phenylmercuric acetate, chlorobutanol, sorbic acid,chlorhexidine, benzyl alcohol, parabens, thimerosal, and mixturesthereof.

Solutions, suspensions, or emulsions for ocular administration may alsocontain one or more excipients known art, such as dispersing agents,wetting agents, and suspending agents.

B. Additional Active Agents

In addition to the sunitinib present in the polymeric particles, theformulation can contain one or more additional therapeutic, diagnostic,and/or prophylactic agents. The active agents can be a small moleculeactive agent or a biomolecule, such as an enzyme or protein,polypeptide, or nucleic acid.

Suitable small molecule active agents include organic and organometalliccompounds. In some instances, the small molecule active agent has amolecular weight of less than about 2000 g/mol, more preferably lessthan about 1500 g/mol, most preferably less than about 1200 g/mol. Thesmall molecule active agent can be a hydrophilic, hydrophobic, oramphiphilic compound.

In some cases, one or more additional active agents may be encapsulatedin, dispersed in, or otherwise associated with the particles. In certainembodiments, one or more additional active agents may also be dissolvedor suspended in the pharmaceutically acceptable carrier.

In the case of pharmaceutical compositions for the treatment of oculardiseases, the formulation may contain one or more ophthalmic drugs. Inparticular embodiments, the ophthalmic drug is a drug used to treat,prevent or diagnose a disease or disorder of the posterior segment eye.Non-limiting examples of ophthalmic drugs include anti-glaucoma agents,anti-angiogenesis agents, anti-infective agents, anti-inflammatoryagents, growth factors, immunosuppressant agents, anti-allergic agents,and combinations thereof.

Representative anti-glaucoma agents include prostaglandin analogs (suchas travoprost, bimatoprost, and latanoprost), beta-andrenergic receptorantagonists (such as timolol, betaxolol, levobetaxolol, and carteolol),alpha-2 adrenergic receptor agonists (such as brimonidine andapraclonidine), carbonic anhydrase inhibitors (such as brinzolamide,acetazolamine, and dorzolamide), miotics (i.e., parasympathomimetics,such as pilocarpine and ecothiopate), seretonergics muscarinics,dopaminergic agonists, and adrenergic agonists (such as apraclonidineand brimonidine).

Representative anti-angiogenesis agents include, but are not limited to,antibodies to vascular endothelial growth factor (VEGF) such asbevacizumab (AVASTIN®) and rhuFAb V2 (ranibizumab, LUCENTIS®), and otheranti-VEGF compounds including aflibercept (EYLEA®); MACUGEN® (pegaptanimsodium, anti-VEGF aptamer or EYE001) (Eyetech Pharmaceuticals); pigmentepithelium derived factor(s) (PEDF); COX-2 inhibitors such as celecoxib(CELEBREX®) and rofecoxib (VIOXX®); interferon alpha; interleukin-12(IL-12); thalidomide (THALOMID®) and derivatives thereof such aslenalidomide (REVLIMID®); squalamine; endostatin; angiostatin; ribozymeinhibitors such as ANGIOZYME® (Sirna Therapeutics); multifunctionalantiangiogenic agents such as NEOVASTAT® (AE-941) (Aeterna Laboratories,Quebec City, Canada); receptor tyrosine kinase (RTK) inhibitors such assunitinib (SUTENT®); tyrosine kinase inhibitors such as sorafenib(Nexavar®) and erlotinib (Tarceva®); antibodies to the epidermal grownfactor receptor such as panitumumab (VECTIBIX®) and cetuximab(ERBITUX®), as well as other anti-angiogenesis agents known in the art.

Anti-infective agents include antiviral agents, antibacterial agents,antiparasitic agents, and anti-fungal agents. Representative antiviralagents include ganciclovir and acyclovir. Representative antibioticagents include aminoglycosides such as streptomycin, amikacin,gentamicin, and tobramycin, ansamycins such as geldanamycin andherbimycin, carbacephems, carbapenems, cephalosporins, glycopeptidessuch as vancomycin, teicoplanin, and telavancin, lincosamides,lipopeptides such as daptomycin, macrolides such as azithromycin,clarithromycin, dirithromycin, and erythromycin, monobactams,nitrofurans, penicillins, polypeptides such as bacitracin, colistin andpolymyxin B, quinolones, sulfonamides, and tetracyclines.

In some cases, the active agent is an anti-allergic agent such asolopatadine and epinastine.

Anti-inflammatory agents include both non-steroidal and steroidalanti-inflammatory agents. Suitable steroidal active agents includeglucocorticoids, progestins, mineralocorticoids, and corticosteroids.

The ophthalmic drug may be present in its neutral form, or in the formof a pharmaceutically acceptable salt. In some cases, it may bedesirable to prepare a formulation containing a salt of an active agentdue to one or more of the salt's advantageous physical properties, suchas enhanced stability or a desirable solubility or dissolution profile.

Generally, pharmaceutically acceptable salts can be prepared by reactionof the free acid or base forms of an active agent with a stoichiometricamount of the appropriate base or acid in water or in an organicsolvent, or in a mixture of the two; generally, non-aqueous media likeether, ethyl acetate, ethanol, isopropanol, or acetonitrile arepreferred. Pharmaceutically acceptable salts include salts of an activeagent derived from inorganic acids, organic acids, alkali metal salts,and alkaline earth metal salts as well as salts formed by reaction ofthe drug with a suitable organic ligand (e.g., quaternary ammoniumsalts). Lists of suitable salts are found, for example, in Remington'sPharmaceutical Sciences, 20th ed., Lippincott Williams & Wilkins,Baltimore, Md., 2000, p. 704. Examples of ophthalmic drugs sometimesadministered in the form of a pharmaceutically acceptable salt includetimolol maleate, brimonidine tartrate, and sodium diclofenac.

In some cases, the active agent is a diagnostic agent imaging orotherwise assessing the eye. Exemplary diagnostic agents includeparamagnetic molecules, fluorescent compounds, magnetic molecules, andradionuclides, x-ray imaging agents, and contrast media.

In certain embodiments, the pharmaceutical composition contains one ormore local anesthetics. Representative local anesthetics includetetracaine, lidocaine, amethocaine, proparacaine, lignocaine, andbupivacaine. In some cases, one or more additional agents, such as ahyaluronidase enzyme, is also added to the formulation to accelerate andimproves dispersal of the local anesthetic.

V. Methods of Use

There are several types of glaucoma. The two main types are open-angleand angle-closure. These are marked by an increase of intraocularpressure (IOP), or pressure inside the eye.

Open-angle glaucoma, the most common form of glaucoma, accounting for atleast 90% of all glaucoma cases, is caused by the slow clogging of thedrainage canals, resulting in increased eye pressure. It has a wide andopen angle between the iris and cornea, develops slowly and is alifelong condition, and has symptoms and damage that are not noticed.“Open-angle” means that the angle where the iris meets the cornea is aswide and open as it should be. Open-angle glaucoma is also calledprimary or chronic glaucoma. It is the most common type of glaucoma,affecting about three million Americans.

Angle-closure glaucoma, a less common form of glaucoma: It is caused byblocked drainage canals, resulting in a sudden rise in intraocularpressure, has a closed or narrow angle between the iris and cornea,develops very quickly, has symptoms and damage that are usually verynoticeable, and demands immediate medical attention. It is also calledacute glaucoma or narrow-angle glaucoma. Unlike open-angle glaucoma,angle-closure glaucoma is a result of the angle between the iris andcornea closing.

Normal-Tension Glaucoma (NTG) or low-tension or normal-pressure glaucomais where the optic nerve is damaged even though the eye pressure is notvery high. Congenital Glaucoma occurs in babies when there is incorrector incomplete development of the eye's drainage canals during theprenatal period. This is a rare condition that may be inherited. Whenuncomplicated, microsurgery can often correct the structural defects.Other cases are treated with medication and surgery. Other Types ofGlaucoma include: Secondary Glaucoma, Pigmentary Glaucoma,Pseudoexfoliative Glaucoma, Traumatic Glaucoma, Neovascular Glaucoma,Irido Corneal Endothelial Syndrome (ICE), and Uveitic Glaucoma.

Open angle glaucoma is the most common form of glaucoma, affecting aboutthree million Americans. It happens when the eye's drainage canalsbecome clogged over time. The inner eye pressure (intraocular pressureor IOP) rises because the correct amount of fluid cannot drain out ofthe eye. With open-angle glaucoma, the entrances to the drainage canalsare clear and should be working correctly. If open-angle glaucoma is notdiagnosed and treated, it can cause a gradual loss of vision. This typeof glaucoma develops slowly and sometimes without noticeable sight lossfor many years. It usually responds well to medication, especially ifcaught early and treated.

Vision loss in glaucoma, a neurodegenerative disease that is the leadingcause of irreversible blindness worldwide, is due to the dysfunction anddeath of retinal ganglion cells (RGCs). Current therapies all act bylowering intraocular pressure (IOP). However, pressure reduction can bedifficult to achieve, and even with significant pressure lowering, RGCloss can continue. Efforts have therefore been made to developneuroprotective agents that would complement IOP-lowering by directlyinhibiting the RGC cell death process, though no neuroprotective agentis yet in clinical use. As described in the examples, using ahigh-content phenotypic screen based on primary RGC cultures, it wasunexpectedly found that the FDA-approved drug sunitinib stronglypromotes RGC survival in rodent glaucoma and traumatic optic neuropathymodels. In order to identify the molecular target(s) through whichsunitinib promotes RGC survival, a high-throughput RNAinterference-based assay was developed, and used to screen the fullmouse kinome. The screen identified dual-leucine zipper kinase (DLK) asa key neuroprotective drug target of sunitinib. Supporting this finding,a number of other neuroprotective kinase inhibitors also inhibit DLK.These include SR8165, axitinib, bosutinib, neratininb, crizotinib,tozasertib, lestautinib, foretinib, TAE-684 and KW-2449.

Although described herein primarily with respect to to sunitinib, it isunderstood that these other compounds could be used in place ofsunitinib. Furthermore, it was shown that DLK undergoes a robustpost-transcriptional upregulation in response to injury that is bothnecessary and sufficient for RGC cell death. Together, the resultsestablish a drug/drug target combination in glaucoma, suggest a possiblebiomarker for RGC injury, and provide a starting point for thedevelopment of more specific neuroprotective DLK inhibitors for thetreatment of glaucoma and other forms of optic nerve disease.

The formulations provide for more sustained controlled release ofsunitinib and similar compounds for reduction or prevention of neuronaldeath due to elevated intraocular pressure associated with glaucoma.Upon administration, the sunitinib or other agents are released over anextended period of time at concentrations which are high enough toproduce therapeutic benefit, but low enough to avoid cytotoxicity.

When administered to the eye, the particles release a low dose of one ormore active agents over an extended period of time, preferably longerthan 3, 7, 10, 15, 21, 25, 30, or 45 days. The structure of the drug ormakeup of the polymeric matrix, particle morphology, and dosage ofparticles administered can be tailored to administer a therapeuticallyeffective amount of one or more active agents to the eye over anextended period of time while minimizing side effects, such as thereduction of scoptopic ERG b-wave amplitudes and/or retinaldegeneration.

Pharmaceutical compositions containing particles for the controlledrelease can be administered to the eye of a patient in need thereof totreat or prevent one or more diseases or disorders of the eye. In somecases, the disease or disorder of the eye affects the posterior segmentof the eye. The posterior segment of the eye, as used herein, refers tothe back two-thirds of the eye, including the anterior hyaloid membraneand all of the optical structures behind it, such as the vitreous humor,retina, choroid, and optic nerve.

1. Methods of Administration

The formulations described herein can be administered locally to the eyeby intravitreal injection (e.g., front, mid or back vitreal injection),subconjunctival injection, intracameral injection, injection into theanterior chamber via the temporal limbus, intrastromal injection,injection into the subchoroidal space, intracorneal injection,subretinal injection, and intraocular injection. In a preferredembodiment, the pharmaceutical composition is administered byintravitreal injection.

The implants described herein can be administered to the eye usingsuitable methods for implantation known in the art. In certainembodiments, the implants are injected intravitreally using a needle,such as a 22-guage needle. Placement of the implant intravitreally maybe varied in view of the implant size, implant shape, and the disease ordisorder to be treated. In some embodiments, the pharmaceuticalcompositions and/or implants described herein are co-administered withone or more additional active agents. “Co-administration”, as usedherein, refers to administration of the controlled release formulationof one or more Drugs with one or more additional active agents withinthe same dosage form, as well as administration using different dosageforms simultaneously or as essentially the same time. “Essentially atthe same time” as used herein generally means within ten minutes,preferably within five minutes, more preferably within two minutes, mostpreferably within in one minute. In some embodiments, the pharmaceuticalcompositions and/or implants described herein are co-administered withone or more additional treatments for a neovascular disease or disorderof the eye. In some embodiments, the pharmaceutical compositions and/orimplants described herein are co-administered with one or moreanti-angiogenesis agent such bevacizumab (AVASTIN®), ranibizumab,LUCENTIS®, or aflibercept (EYLEA®).

b. Dosage

Preferably, the particles will release an effective amount of sunitinibover an extended period of time. In preferred embodiments, the particlesrelease an effective amount of sunitinib over a period of at least twoweeks, more preferably over a period of at least four weeks, morepreferably over a period of at least six weeks, most preferably over aperiod of at least eight weeks. In some embodiments, the particlesrelease an effective amount of sunitinib over a period of three monthsor longer to reduce or prevent neuronal cell death, especially that ofthe optic nerve due to elevated intraocular pressure.

Although described with reference to reference to the eye, it isunderstood that the formulation may be administered to other sites toprotect neuronal cells, including the brain.

The present invention will be further understood by reference to thefollowing non-limiting examples.

EXAMPLE 1. PREPARATION OF SUNITINIB-ENCAPSULATED MICROPARTICLES AND INVITRO CHARACTERIZATIONS Materials and Methods Preparation ofSunitinib-Encapsulated Microparticles

Polymer microparticles of PLGA and/or a diblock copolymer of PLGA andPEG covalently conjugated to PLGA (M_(w) 45 kDa) (PLGA45k-PEG5k) with orwithout sunitinib malate were prepared using a single emulsion solventevaporation method. Briefly, PLGA and/or PLGA-PEG were first dissolvedin dichloromethane (DCM) and sunitinib malate was dissolved in dimethylsulfoxide (DMSO) at predetermined concentrations. The polymer solutionand the drug solution were mixed to form a homogeneous solution (organicphase). The organic phase was added to an aqueous solution of 1%polyvinyl alcohol (PVA) (Polysciences, Mw 25 kDa, 88% hydroplyzed) andhomogenized at 5,000 rpm for 1 min using an L5M-A laboratory mixer(Silverson Machines Inc., East Longmeadow, Mass.) to obtain an emulsion.

The solvent-laden microparticles in the emulsion was then hardened bystirring at room temperature for >2 hr to allow the DCM to evaporate.The microparticles were collected by sedimentation and centrifugation,washed three times in water and dried by lyophilization.

Both cationic and ionic surfactants, Sodium dodecyl sulfate (“SDS”) andHexadecyltrimethylammonium (“HDTA”), were added to the solvents to makethe particles. These were substituted with a non-ionic solvent,polyvinyl alcohol (“PVA”). Sunitinib free base crystallized and couldnot be utilized. DMSO was added to the solvent, alone and in combinationwith surfactant.

The particles were collected by centrifugation, washed, and freeze-driedto a powder to be reconstituted prior to administration. Averageparticle size and size distribution was determined using a CoulterMultisizer. Drug release kinetics in infinite sink conditions at 37° C.in PBS and vitreous solution mimic were determined.

Determination of Drug Loading

Drug loading was determined by UV-Vis spectrophotometry. Microparticlescontaining sunitinib (10 mg total weight) were dissolved in anhydrousDMSO (1 mL) and further diluted until the concentration of drug was inthe linear range of the standard curve of UV absorbance of the drug. Theconcentration of the drug was determined by comparing the UV absorbanceto a standard curve. Drug loading is defined as the weight ratio of drugto microparticles.

In Vitro Drug Release

Microparticles containing sunitinib (10 mg total weight) were suspendedin 4 mL of PBS containing 1% TWEEN® 20 in a 6-mL glass vial andincubated at 37° C. under shaking at 150 rpm. At predetermined timepoints, 3 mL of the supernatant was withdrawn after particles settled tothe bottom of the vial and replaced with 3 mL of fresh release medium.The drug content in the supernatant was determined by UV-Visspectrophotometry or HPLC.

Measurement of Average Size and Size Distribution of Microparticles

Several milligrams of the microparticles were first suspended in waterand dispersed in an ISOTON® diluent. The mean particle size anddistributions were determined using a COULTER MULTISIZER IV (BeckmanCoulter, Inc., Brea, Calif.).

Results

Both cationic and ionic surfactants, such as Sodium dodecyl sulfate(“SDS”) and Hexadecyltrimethylammonium (“HDTA”), were added to thesolvents to make the particles. Loadings were extremely low (0.20% withSDS and 0.27% with HDTA). Substituting a non-ionic solvent such aspolyvinyl alcohol (“PVA”) increased loading, up to 1.1%. Sunitinib freebase crystallized and could not be utilized to obtain higher loading.Using DMSO increased loading even more so, up to around 5%.

TABLE 1 Composition and process parameters of sunitinib-encapsulatedpolymer microparticle formulations Organic phase PLGA 5050-PEG Encap-Formu- 5kD (10% Sunitinib Aqueous phase Emulsion Drug Target sulationlation PLGA PLGA PEG by DCM malate DMSO Volume rate loading loadingefficiency ID (mg) type wt) (mg) (mL) (mg) (mL) Surfactant (mL) (rpm)(wt %) (wt %) (%) DC-2-55-2 560 7525 4A 5.6 4 90 2 1% PVA in 200 50003.1 13.7 22.6 pH 4 buffer DC-2-55-3 560 7525 4A 5.6 4 90 2 1% Borate 2005000 NA 13.7 NA Buffer (pH 10) DC-2-55-4 560 7525 4A 5.6 4 90 2 1% PVAin 200 5000 5.0 13.7 36.4 H2O (pH 6) DC-2-55-5 560 7525 4A 5.6 4 90 2 1%PVA in 200 5000 11.5 13.7 83.8 PBS (pH 7.4) DC-2-50-1 800 7525 4A 8 4145 2 1% PVA in PBS 200 5000 13.9 15.2 91.4 DC-2-50-2 560 7525 4A 5.6 4100 2 1% PVA in PBS 200 5000 12 15.0 79.9 DC-2-50-3 400 7525 4A 4 4 70 21% PVA in PBS 200 5000 7.8 14.8 52.8 DC-2-50-4 280 7525 4A 2.8 4 50 2 1%PVA in PBS 200 5000 6.8 15.0 45.3 DC-2-50-5 200 7525 4A 2 4 35 2 1% PVAin PBS 200 5000 4.9 14.8 33.2 DC-1-53-1 400 7525 6E 4 4 160 2 1% PVA inPBS 200 5000 23.7 28.4 83.6 DC-1-53-2 400 8515 6E 4 4 160 2 1% PVA inPBS 200 5000 23.9 28.4 84.1 DC-1-53-3 400 8515 6A 4 4 160 2 1% PVA inPBS 200 5000 22.6 28.4 79.6 JCK-1-72-1 400 5050 2A 8 80 2 1% PVA in H2O200 4000 3.4 16.7 20.1 YY-1-59-1 200 7525 4A/ 2 3 40 1 1% PVA in PBS 1004000 6.8 16.5 41.2 7525 1.5A (1:1) YY-1-83-1 554 7525 4A 6 4 160 2 1%PVA in PBS 200 5000 20.5 22.2 92.2 YY-1-83-2 504 7525 4A 56 4 160 2 1%PVA in PBS 200 5000 20.2 22.2 90.7 YY-1-93-1 400 7525 4A 4 4 90 2 1% PVAin PBS 200 4000 12.4 18.2 68.1 YY-1-93-2 560 7525 4A 5.6 4 90 2 1% PVAin PBS 200 5000 11.6 13.7 84.5 YY-1-96-1 2240 7525 4A 22.4 16 360 8 1%PVA in PBS 800 3000 10.1 13.7 73.6 JCK-1-26-8 100 7525 6E 2 75 1 1% PVAin PBS 100 4000 34.5 42.9 80.5

EXAMPLE 2. THE EFFECT OF AQUEOUS PH ON ENCAPSULATION EFFICIENCY OFSUNITINIB Materials and Methods

As the solubility of sunitinib in aqueous solution was shown to be pHdependent, microparticle formulations encapsulating sunitinib wereprepared in aqueous phases of various pH values to investigate theeffect of aqueous pH on drug encapsulation.

Results

As shown in Table 2, the loading and encapsulation efficiency increasedsignificantly when the aqueous pH was increased from 4 to 7.4. However,when the pH was adjusted to 10 and the aqueous solution became morebasic, the morphology of many particles changed from spherical toirregular shapes and some particles formed aggregates, suggestingaqueous solution of high pH is also unfavorable for producing particlesof high loading of sunitinib and high quality.

TABLE 2 The effect of aqueous phase pH on encapsulation efficiency ofsunitinib Mean Aqueous Actual Target Encapsulation diameter Sample ID pHloading loading efficiency (μm) DC-2-55-2 4  3.1% 13.7% 22% 27.0 ± 7.9DC-2-55-4 6  5.0% 13.7% 36% 28.0 ± 8.3 DC-2-55-5 7.4 11.5% 13.7% 84%27.4 ± 7.6 DC-2-55-3 10 NA 13.7% NA NA

EXAMPLE 3: TESTING OF SUNITINIB FOR GLAUCOMA TREATMENT Materials andMethods Reagents

Sunitinib, lestaurtinib, crizotinib, axitinib, bosutinib, imatinib,tandutinib, vandetanib, sorafenib and vatalanib were purchased from LClabs, Forentinib, KW-2449 from Selleckchem, and tozasertib fromBiovision life science.

PLGA Microspheres

Polymer microparticles loaded with sunitinib, SR8165, or tozasertib wereprepared using a single emulsion solvent evaporation method. Briefly, asolution was made by mixing 200 mg of poly(lactic-co-glycolic acid)(PLGA 50:50, 2A, 0.15-0.25 dL/g, MW15K-17K, Lakeshore Biomaterials,Birmingham, Ala.) dissolved in 4 mL methylene chloride with one ofvarious drugs dissolved in DMSO (40 mg sunitinib in 1 mL DMSO, 40 mgSR8165 in 0.5 mL DMSO, 40 mg tozasertib in 1 mL DMSO). The mixture washomogenized at 4000 rpm (Silverson Homogenizer, model L4RT, CheshamBucks, England) for 1 min into an aqueous solution containing 1%polyvinyl alcohol (PVA, MW=25 KDa, Polysciences, Warrington, Pa.). Theparticles were then stirred for 2 hours to allow hardening, collected bycentrifugation at 5000 g for 5 minutes, washed with deionized water 3times, and freeze-dried to a powder that could be reconstituted prior toadministration. Microparticle size was determined using a CoulterMultisizer IIe (Beckman-Coulter Inc., Fullerton, Calif.). To determinethe drug release rate in vitro, 5 mg of drug-loaded particles weresuspended in 2 mL of phosphate-buffered saline (pH 7.4) and incubated at37° C. on a rotator. At selected time points, microparticles wereprecipitated by centrifugation, and the supernatant removed and replacedwith 2 mL of fresh phosphate buffer. Supernatants were analyzed byspectrophotometry at 420 nm for sunitinib and SR8165, and 252 nm fortozasertib.

Statistical Analysis

All statistical analyses were performed with the unpairedMann-Whitney-Wilcoxon test.

RGC Purification, Culture, Screening and Imaging

All animal use was in accordance with ARVO Statement for the Use ofAnimals, and all the experimental procedures were performed incompliance with animal protocols approved by the IACUC at Johns HopkinsUniversity. Retinas were isolated from postnatal 0-5 day mice anddissociated with papain. Microglia was immunodepleted with anti-CD11bconjugated Dynabeads. The suspension of retinal cells were immunopannedon plates pre-conjugated with anti-Thy1.2 antibody (Serotec, MCA028) andanti-mouse IgM at room temperature (RT). After washing, RGCs werereleased from the plate by a cell lifter, counted, and seeded at adensity of 10,000 per well in 96-well plates in the media composed ofNeurobasal, B27, N2 supplement, L-glutamine, andpenicillin/streptomycin. After a 72 hour culture at 37° C., RGCs werestained with calcein AM, ethidium homodimer, and Hoechst 33342. Imageswere taken from portions of each well with Cellomics Kinetscan, and cellsurvival was quantified and calculated with the algorithms in CellomicsNeuroprofiling package. As indicated, RGC viability was alternativelymeasured by CellTiter-Glo luminescence (Promega).

For siRNA-based screening, the siRNAs from the Sigma Mission MouseKinome library were complexed with NeuroMag (Oz Biosciences) at a finalconcentration of 20 nM. RGCs were then reverse transfected on astationary magnet and assayed for survival 72 hours later.Oligonucleotides conferring survival more than 3 SD from thenontargeting siRNAs were considered neuroprotective (106 siRNA, 5.4%).Confirmatory siRNAs were obtained from both Dharmacon and Ambion. Forsmall molecule-based screening, serially diluted compounds in DMSO weretransferred to 1536 well assay plates by a 23 nL pintool array(Kalypsys, San Diego, Calif.), with a final concentration of 0.057% DMSOfor each respective compound concentration. RGCs were cultured for 48 h,and cell viability was analyzed on a plate reader (ViewLux, PerkinElmer) using the bioluminescent CellTiterGlo (Promega) assay.Concentration response curves were created using CurveFit (NIH NCATS).The screened libraries include the modified Tocriscreen (1395 compounds)collection, FDA-approved drugs (2814 compounds), LOPAC (1208 compounds)and PTL2/PTL3 (based on pteridin, pyrimidine and quinazoline scaffolds;2319 compounds).

Rat Intravitreal Injections

6-week old male Wistar rats were anesthetized with ketamine/xylazine. Apartial periotomy was made to expose the sclera. The injection site wasapproximately 1 mm posterior to the ora serrata, and the injection glasspipet was angled towards the optic disc in order to avoid lens injury. 5μL (10 μg) of PLGA microspheres were injected with a glass pipet andHamilton syringe.

Rat Optic Nerve Transection

The optic nerve was exposed by a partial peritomy and intraorbitaldissection of the extraocular muscles, and then transected with a25-gauge needle. 4-Di-10-ASP were then applied to the proximal nervestump. Care was taken to avoid vascular injury during the transection,and retinal perfusion was examined after nerve transection. Two weeksafter transection, rats were sacrificed and enucleated. Retinas wereflatmounted, imaged with confocal microscopy and the number of4-Di-10-ASP-labeled cells with RGC morphology was quantified Imaging andquantification of RGC survival were performed in a masked fashion.

Rat Laser-Induced Ocular Hypertension

Intraocular pressure (IOP) was unilaterally elevated by laser treatmentof the trabecular meshwork as previously described. Briefly, 6-week oldWistar male rats were anesthetized with ketamine/xylazine. On twoconsecutive weeks, 40-50 532 nm diode laser spots were applied to theprelimbal region (50 μm diameter, 600 mW power and 0.6 secondsduration). Under anesthesia, the IOP of laser-treated and fellow eyeswas measured with TonoLab one and three days after laser treatment. Fourweeks following laser treatment, rats were perfused with 4%paraformaldehyde in phosphate buffer. Optic nerves were isolated,postfixed with 1% osmium tetroxide, embedded in epoxy resin and stainedwith 1% toluidine blue. Images from 10 random and nonoverlapping fieldswere taken with 100× oil phase contrast objective. The area of entireoptic nerve cross-sections were imaged with 10× magnification, and usedwith axon counts from the 10 field to derive axon counts per nerve. Thelaser treatment and acquisition of optic nerve images were performed ina masked fashion.

Mouse Intravitreal Injection and Optic Nerve Crush

3-month old male C57BL/6 and Dlk floxed mice (BL/6 background) wereanesthetized with ketamine/xylazine and intravitreally injected with10¹⁰ DNA-containing particles of capsid-mutant (Y444, 500, 730F) AAV2expressing Cre recombinase from the chicken β-actin promoter. 7 dayslater, optic nerve was surgically exposed and crushed with Dumont N5self-closing forceps 1 mm behind the globe for 3 seconds. 10 daysfollowing nerve crush, eyes were enucleated, fixed and RGC survival wasmeasured with flatmount immunostaining for βIII-tubulin and Brn3.Intravitreal injection, optic nerve crush, immunofluorescence and RGCcounting were performed in a masked fashion.

Western Blots, Immunofluorescence and RT-PCR

Western blots were performed according to the standard protocol. Thefollowing antibodies were from Cell Signaling Technology: Phospho-JNK,Thr183/Tyr185 (4671); JNK (9258); Phospho-MKK7 (4171), and MKK7 (4172).Monoclonal anti-alpha-tubulin antibody (T6074) was purchased from Sigma.DLK rabbit polyclonal was provided by S. Hirai.

Retinal immunofluorescence was performed following standard protocols.The following antibodies were used: mouse neuronal class βIII tubulin(clone TUJ1, 1:500, Covance), rabbit polyclonal anti-DLK (1:200, S.Hirai), and goat polyclonal Brn3 (C-13, 1:100), rabbit anti-Cre (1:100,Novus).

Dlk mRNA levels were measured with RT-PCR with the following primer set:

primer set: (SEQ ID NO: 1) 5′-ATTCCTCAGCCATCATCTGG-3′ and (SEQ ID NO: 2)5′-ATTTCGTGGTTTGCTGTTCC-3′.

Electrophysiology

Recordings were made by using the whole-cell patch-clamp technique inboth current- and voltage-clamp modes with an Axopatch 200B. Data werelow-pass filtered at 1 kHz (Bessel) and sampled at 10 kHz. A liquidjunction potential of −2 mV has been corrected, and the restingpotential was estimated to be −62±2.2 mV (Mean±SEM, n=13). The recordingpipette was filled with the following intracellular solution (in mM):100 K-gluconate, 50 KCl, 20 HEPES, 10 EGTA, 5 MgCl₂, 2 ATP, 0.1 GTP, pHadjusted to 7.33 with KOH. The cells were continuously perfused with (inmM): 140 NaCl, 5 KCl, 1 MgCl₂, 2.5 CaCl₂, 10 glucose, 10 HEPES, pH 7.4with NaOH.

Production of AAV Vectors

AAV vector preparations are produced by the 2-plasmid, co-transfectionmethod with modifications²³. Briefly, ˜10⁹ HEK 293 cells is cultured inDMEM with 5% fetal bovine serum and antibiotics. DNA transfection of thetwo vector plasmids by CaPO4 precipitation then is allowed to incubateat 37° C. in 7% CO₂ for 60 h. The cells are then harvested, lysed bythree freeze/thaw cycles, the crude lysate clarified by centrifugationand the resulting vector-containing supernatant run on a discontinuousiodixanol step gradient. The vector-containing fraction is furtherpurified and concentrated by column chromatography on a 5-ml HiTrap QSepharose column using a Pharmacia AKTA FPLC system. The vector iseluted from the column using 215 mM NaCl, pH 8.0, and the AAV peakcollected, concentrated and buffer exchanged into Alcon BSS with 0.014%Tween 20. Before release, vector purity is assessed by silver-stainedSDS-PAGE, a negative bioburden test, and an endotoxin test in theacceptable range. Finally, vector is titered for DNase-resistant vectorgenomes by Real-Time PCR relative to a reference AAV vector standard.

Results High-Content, High-Throughput, Phenotypic Screen

Screening over 6,000 unique compounds at multiple doses⁵ repeatedlyidentified sunitinib and related oxindole analogs as being highlyneuroprotective. Sunitinib treatment led to a dose-dependent increase inthe viability of primary RGCs, with maximal activity between 0.5 and 1μM (FIG. 1A). Increased survival was associated with a correspondingdecrease in markers of apoptosis, including caspase activation, nuclearcondensation and fragmentation.

Rescue of RGC Death in Response to Optic Nerve Transection

The ability of sunitinib to rescue RGC death in vivo in response tooptic nerve transection was tested. Sunitinib, or its vehicle control,were packaged in poly(lactic-co-glycolic acid) (PLGA)-based,slow-eluting microspheres and injected intravitreally into Wistar rats.Seven days later, optic nerves were transected and RGCs wereretrogradely-labeled by applying 4-Di-10-ASP to the proximal nervestump. At two weeks post-transection, sunitinib-treated compared tocontrol animals showed a 3-4 fold increase in surviving RGCs (FIG. 1B).To evaluate sunitinib's neuroprotective activity in a glaucoma model,rats were pretreated with intravitreal vehicle- or sunitinib-elutingmicrospheres and then used diode laser treatment of the trabecularmeshwork to increase IOP (FIGS. 2A-2C). In eyes injected with controlmicrospheres, there was a 58% reduction in optic nerve axons at onemonth. However, in eyes treated with sunitinib-eluting microspheres,axon loss was reduced by 40% (p<0.05, FIG. 1C). SR8165, asunitinib-analog found to have greater efficacy in vitro (FIG. 5A),conferred similar neuroprotection upon delivery from PLGA microspheres(FIG. 1C). Together, these results establish sunitinib, and relatedoxindole analogs, as neuroprotective agents for glaucoma.

To confirm that kinase inhibition mediated the neuroprotective activityof these oxindole kinase inhibitors, the activity of SU6656, aneuroprotective analog of sunitinib, was compared to that of SR8020, anotherwise identical derivative in which a hydrogen to methyl groupsubstitution is predicted to disrupt the binding to the kinaseATP-binding pocket. SR8020 failed to show survival-promoting activity,thus suggesting that the neuroprotective activity of sunitinib and itsanalogs is mediated through ATP-competitive kinase inhibition. Atneuroprotective concentrations, sunitinib inhibits nearly 200 kinases.

TABLE 3 Particle Size, Loading, Release Drug Loading Drug Release RateParticle Size (wt. drug/ (μg drug/ a (μm) total wt.) mg particles)Sunitinib  9.1 ± 1.4 11.0 2.5 SR8165 10.4 ± 1.7 11.0 6.5 Tozasertib 11.2± 1.5 5.5 0.6

FIG. 3A is a graph of viable cells showing survival of immunopannedRGCs, treated with increasing doses of SR8165, after 72 hours inculture. The most efficacious doses of sunitinib are shown forcomparison. FIGS. 3B-3F are graphs of viable cells, showing the lack ofneuroprotective activity of kinase inhibitors targeting VEGFR2, c-Kit,FLT3 and PDGFRs, of immunopanned RGCs, treated with increasing doses ofthe various kinase inhibitors, after 72 hours in culture.

Among the kinases potently inhibited are vascular endothelial growthfactor receptor 2 (VEGFR2), c-Kit, FLT3 and platelet-derived growthfactor receptors (PDGFRs). However, other small molecules known toinhibit one or more of those same receptor tyrosine kinases, includingimatinib, sorafenib, tandutinib, vandetanib, and vatalanib, all lackneuroprotective activity (FIGS. 3B-3F). These results, together with therelatively high concentrations required for neuroprotection in vitro,suggested that the kinase(s) whose inhibition promotes RGC survival areone or more low-affinity targets of sunitinib. In order to identify therelevant kinase(s), the primary RGC platform was adapted for an unbiasedRNA interference-based screen of the entire mouse kinome. It wasreasoned that kinases, whose knockdown increased RGC survival, wouldrepresent possible relevant targets for neuroprotective kinaseinhibition. Since traditional transfection procedures resulted inminimal RGC transfection, or were toxic, a magnetic nanoparticle-basedhigh-throughput method was developed that provided efficient siRNAdelivery to cultured primary RGCs. An arrayed library of 1869 siRNAs wasthen screened against 623 kinases, providing three-fold coverage of themouse kinome.

The only two kinases for which all three siRNAs were significantlyneuroprotective were mitogen-activated protein kinase kinase kinase12/dual-leucine zipper kinase (Map3k12/Dlk) and its only knownsubstrate, mitogen-activated protein kinase kinase 7 (Map2k7/Mkk7).Involvement of both kinases was subsequently confirmed in secondaryscreening using an independent set of siRNAs. MKK7 and its homolog,MKK4, are the canonical activators of the c-Jun N-terminal kinases(JNK1-3), key mediators of RGC cell death. The results indicate that DLKmay be the as-yet-unidentified trigger for JNK activation and cell deathin RGCs. Indeed, DLK has been shown to mediate developmental apoptosisin peripheral motor and sensory neurons, but no role in adult CNSneurodegenerations has been firmly established.

To explore the role of DLK in mediating RGC death, immunopanned RGCswere transfected with DLK siRNA, or a nontargeting control, and survivalover time followed. As predicted, DLK siRNA knocked down the level ofDLK mRNA and protein and inhibited phosphorylation of JNK, a marker ofactivation of JNK downstream signaling (FIG. 4A). While nontargetingsiRNA-transfected cells were dead by 72 hours, RGCs transfected with DLKsiRNA survived for greater than 3 weeks (FIG. 4B). To determine whetherthe RGCs that are kept alive for extended periods with DLK siRNA orsunitinib treatment remain functional, patch-clamp recordings wereperformed at two weeks in culture. Consistent with persistentfunctionality, the RGCs conducted action potentials in response todepolarizing current and were responsive to exogenously appliedglutamate. To test the role of DLK in vivo in response to axonal injury,mice carrying a floxed allele of Dlk¹ with capsid-modifiedadeno-associated virus 2 (AAV2) expressing the P1 bacteriophagerecombinase Cre were intravitreally injected. After sufficient time forCre-mediated deletion of Dlk, eyes were subjected to optic nerve crush.Compared to either Dlk^(+/+) mice injected with AAV2-Cre or Dlk^(fl/fl)mice in the absence of Cre, Dlk^(fl/fl) mice injected with AAV2-Cre hada 75% reduction in RGC loss (FIGS. 4C and 4D).

As DLK appears to be a critical mediator of RGC cell death in vitro andin vivo, the mechanism of DLK regulation was examined Surprisingly, andunlike other members of the JNK cascade, DLK protein is undetectable inuninjured RGCs both in vitro and in vivo (FIG. 5A). However, afterimmunopanning in vitro (when RGCs are necessarily axotomized andinjured), optic nerve crush, or transection in vivo, there is a robustupregulation of DLK protein). In contrast, Dlk transcript levelsremained relatively constant after injury (FIG. 5A), indicatingincreased translation and/or decreased protein turnover as the mechanismmediating DLK upregulation.

To directly test the hypothesis that increased DLK protein can triggerRGC cell death, adenovirus was used to overexpress GFP, DLK or akinase-dead (KD) version of DLK (K185R). Primary RGCs were infected andsurvival measured 48 hours later. Consistent with the model, wildtypeDLK overexpression hastened cell death, while overexpression of K185RDLK functioned as a dominant-negative, as assessed by JNKphosphorylation, and actually increased survival (FIG. 5B).

Given that both sunitinib treatment and DLK knockdown promote RGCsurvival, sunitinib's neuroprotective activity was mediated, at least inpart, by DLK pathway inhibition. To assess sunitinib's effect on DLKsignaling in RGCs, immunopanned cells were cultured in the presence ofincreasing amounts of sunitinib. The same concentrations that increaseRGC survival caused a decrease in the phosphorylation of targetsdownstream of DLK, including MKK7 and JNK.

SR8165, a sunitinib analog with a widened therapeutic window, reducedDLK toxicity. Nine other compounds reported to bind DLK (axitinib,bosutinib, neratininb, crizotinib, tozasertib, lestautinib, foretinib,TAE-684 and KW-2449) were tested. Except for neratinib and TAE-684,which were limited by toxicity at nanomolar doses, the remaining kinaseinhibitors all promoted the survival of primary RGCs in culture, withneuroprotective doses that roughly correlated with their biochemicalaffinity for purified DLK (FIGS. 6A-6H). To confirm these findings invivo, intravitreal tozasertib in a slow release formulation was tested,and found that it protected RGCs in both the optic nerve transection andglaucoma models (FIGS. 6I and 6J).

The high-throughput screening identified sunitinib as a novelneuroprotective agent capable of promoting RGC survival in vitro and invivo, including in a rodent model of glaucoma. Although initially aparadoxical finding, given the drug's inhibition of growth receptorsignaling and stimulation of apoptosis, the finding that sunitinib'sneuroprotective activity is likely mediated through inhibition of JNKsignaling, via the DLK pathway, provides a mechanistic explanation forits neuroprotective activity. These results establish a therapeuticstrategy for the treatment of glaucoma and related optic neuropathies,and may also have relevance to other CNS neurodegenerations.

Modifications and variations of the sunitinib formulations and methodsof use thereof will be apparent to those of skill in the art and areintended to come within the scope of the appended claims.

1-9. (canceled)
 10. A composition for administration to the eyecomprising polymeric microparticles with increased sunitinibencapsulation having an average diameter between one and 50 micronscomprising sunitinib or a pharmaceutically acceptable salt thereofencapsulated in a blend of poly(lactide-co-glycolide) (PLGA) andpoly(lactide-co-glycolide) (PLGA) conjugated to polyethylene glycol(PEG), wherein the polymeric microparticles release the sunitinib for atleast two weeks and wherein the polymeric microparticles comprisegreater than 5% sunitinib.
 11. The composition of claim 10, wherein themicroparticles further comprise PLA.
 12. The composition of claim 10,wherein the pharmaceutically acceptable salt is sunitinib malate. 13.The composition of claim 10, wherein the average diameter of themicroparticles is between one and 30 microns.
 14. The composition ofclaim 10, wherein the polymeric microparticles are administered viaintravitreal injection.
 15. The composition of claim 10, wherein thepolymeric microparticles are administered via subconjunctival injection.16. The composition of claim 10, wherein the polymeric microparticlescomprise greater than 10% sunitinib.
 17. The composition of claim 10,wherein the polymeric microparticles comprise greater than 15%sunitinib.